Cells of higher vertebrates have evolved mechanisms enabling them to display on their surfaces a sampling of their intracellular contents, in the form of peptides bound to major histocompatibility complex (MHC) class I molecules. Such complexes. are required not only for the recognition and destruction of cells harboring intracellular pathogens, or cells containing altered forms of self proteins, such as oncogenes, but also for the positive and negative selection of T cells during their development in the thymus. It is therefore important to determine how these peptide-MHC complexes are generated. Moreover, recent data suggest that not all peptides of a given antigen which can bind to a particular MHC class I molecule are in fact produced under normal circumstances, and that polymorphism in antigen processing may alter the repertoire of peptides presented to T cells in different individuals. Thus, it is important to determine whether and to what extent the specificity of, and potential polymorphism in, their generation affects the repertoire of peptides that are presented to T cells. Despite their critical importance, the mechanisms by which peptides relevant to presentation to T cells are produced in normal cells are currently unknown. However, recent evidence implicates a large intracellular structure called the LMP complex in this process. Although the circumstantial evidence is strong, no direct proof for this function yet exists. The experiments described in this proposal are designed to test this hypothesis both genetically and functionally. Specifically, we will determine whether LMP complex genes are required for normal antigen processing, and whether purified LMP complex is capable of producing relevant peptides in vitro. Furthermore, the effect of the known (allelic) structural variation in this complex on its proteolytic activity and/or specificity will be determined. We will also expand on the preliminary indications that different forms of the complex exist in different tissues, and test the potential relationship of this structural variation to function. The results of these studies may have important implications for individual differences in immune responsiveness, the genetic basis of MHC-linked predisposition to disease, and MHC-class I matched and non-matched transplantation.